WO2004000274A1

WO2004000274A1 - Oral delivery of biological active agents in a nanoemulsion formulation to the human body

Info

Publication number: WO2004000274A1
Application number: PCT/HU2003/000049
Authority: WO
Inventors: Endre D. Radics
Original assignee: Radics Endre D
Priority date: 2002-06-19
Filing date: 2003-06-18
Publication date: 2003-12-31
Also published as: GB0500594D0; HUP0202032A2; AU2003244885A1; HU0202032D0

Abstract

Present invention relates to the field of compositions of biologically active agents (nutritional supplements, drugs and pharmaceutical agents), their dosage form and new method of administration into the human body. The invention combines the idea of application of nanoemulsions as transport systems for agents and the oral delivery method of said compositions through absorption through the buccal mucosa. Said nanoemulsions contain both water-soluble and fat-soluble biologically active agents, which do not need the conventional emulsifier additives in order to show physical stability. The phase separation of the fat and water soluble particles of the solution is avoided by the addition of pure natural phospholipids. The nanoemulsions with mean droplet diameters ranging from 0,00005 to 0,0001 mm are dispersed by a mechanical pump spray into the oral cavity, where they are absorbed through the buccal mucosa and reach the blood stream and benefit the body.

Description

ORAL DELIVERY OF BIOLOGICAL ACTIVE AGENTS IN A NANOEMULSION FORMULATION TO THE HUMAN BODY

SUBJECT AND TECHNICAL FIELD OF THE INVENTION

Present invention relates to the field of compositions of biologically active agents (nutritional supplements, drugs and pharmaceutical agents), their dosage form and new method of administration into the human body. The invention combines the idea of application of nanoemulsions as transport systems for agents and the oral delivery method of said compositions through absorption through the buccal mucosa.

Said nanoemulsions contain both water-soluble and fat-soluble biologically active agents, which do not need the conventional emulsifier additives in order to show physical stability. The phase separation of the fat and water soluble particles of the solution is avoided by the addition of pure natural phospholipids.

The nanoemulsions with mean droplet diameters ranging from 0,00005 to 0,0001 mm are dispersed by a mechanical pump spray into the oral cavity, where they are absorbed through the buccal mucosa and reach the blood stream and benefit the body.

Advantages offered by the invention

By application of the delivery method based on the absorption through the buccal mucosa, the digestive system is bypassed: biologically active agents reach the blood stream directly from the buccal mucosa. Therefore

1. the administered agents show their effect sooner ( H-l minutes, against hour to 2 hours in case of delivery through the digestive system) ;

2. the biologically active agents are absorbed in greater percentage (95-98%), than in conventional delivery forms (pills gen. ca. 10%, capsules gen. ca. 15-20%, sublingual liquids gen. ca. 30%, etc.) and 3. the delivery of biologically active agents is executed on a user- friendly and convenient way.

Through application of the nanoemulsion

1. administration of both water-soluble and fat-soluble agents is possible;

2. a physically stable mixture of water-soluble and fat-soluble biologically active agents is achieved and maintained without addition of harmful, synthetic emulsifiers;

3. the particles that are enclosed by a phospholipid layer show greater resistance against the degradation effects of the enzymes of the saliva;

4. the phospholipid enclosure enables an active transport: the agents can access the cells faster and in greater percentage;

5. if needed, a delayed effect can be achieved through addition of polymer layers (drugs).

Components of the invention

The invention is a new oral delivery method for administration of biologically active agents, especially vitamins, minerals, amino acids, herbs and plant extracts, as well as pharmaceutical agents, drugs and hormones into the human body. The invention relates to the delivery method, the form of the biologically active solution and the composition of the active ingredients:

1. Method: Mechanical pump activated oral spray with a dispenser hole of max. 50 micron diameter. The agents sprayed into the oral cavity are absorbed through the buccal mucosa and reach the blood stream.

2. Form: The administered agents are transported in a nanoemulsion formulation, which nanoemulsion can contain both fat-soluble and water- soluble components and shows stability without addition of conventional, synthetic emulsifiers.

3. Composition: the administered solution can contain, but is not limited to one or more of the following ingredients:

- Vitamins: both water-soluble and fat-soluble, especially the fat soluble vitamins A, E, D, and K,

- Minerals, especially calcium, magnesium and chrome, Amino acids, Coenzyme Q10,

- Herbs and plant extracts, especially gingko biloba, ginseng, grape seed and pine bark proanthocyanidin extract, echinacea

- pharmaceutical agents, drugs, such as glucosarαine sulfate, chondroitin sulfate,

- hormones, such as melatonin, DHEA

BACKGROUND ART, TECHNICAL STATE

Method of absorption through the buccal mucosa

Nutritional supplements and drugs, pharmaceutical agents are typically provided in solid dosage formulations that are taken orally. Examples include coated and compressed pills, compressed capsules or two piece gelatine capsules. These products have the advantage, that they are easy and relatively inexpensive to produce, largely administered and quite stable. However their effectiveness depends on to which amount they can be absorbed and benefit the human body.

Solid formulations have the disadvantage that most of the ingredients are degraded by stomach acids. Degradation reduces the efficiency and/or therapeutical effects of the ingredients administered in solid forms.

An additional disadvantage of products in solid forms is that a part of humans has difficulties to swallow them. Swallowing a pill causes problems to children, to elder generations and to a high percentage of the adult population. Further, the gag reflex action limits the size of the pills and capsules: the larger the pill, the higher percentage of the population is unable to swallow it. This problem becomes highly significant when high dosages are needed to be administered just in order to overcome the degradation effect of the stomach acids.

The effectiveness of biologically active agents is largely dependent on the ratio and amount of the agents that reach the blood stream. In certain cases, the rapidity is also significant. The better the absorption ratio, the smaller dosage is needed to be administered and the more accurate the administration can be. Absorption of products in solid dosage form happens in two steps:

1. dissolution

2. absorption

This two-step process reduces the absorption ratio and slows the effectiveness. Direct delivery to the veins, to the muscle tissue and under the skin go far beyond the solid oral dosage forms in effectiveness, however due to their hospital character (injection) they are ineligible for everyday civil mass application.

Through the past few decades science has searched for and found more effective and simple delivery methods for administering biologically active agents to the human body.

Liquids have proven themselves more effective than solid dosage forms because they do not need to dissolve and therefore bypass the first step of the usual absorption process of solid forms in the digestive system, however they are considerably degraded by digestive acids .

The skin patch applied the method of direct absorption through the skin, however it brought up the problem of getting the ingredients into the deeper layers of the skin. The wax-like fat tissue of the epidermis prevents the penetration of many agents into the skin. The lipid and keratin containing structure of the horny tissue creates a biological defence system, therefore ingredients can hardly get through dense skin surfaces .

During the last ten years more and more scientific publications dealt with the application of the mucous membranes, especially the buccal mucosa as absorption area. The highly vascularized mucosa of the mouth allows agents a fast and great absorption.

The sublingual nitro-glycerine tablet is the most common example for application of the oral absorption method through the buccal mucosa. When needed, heart patients can quickly get the active ingredient to the heart with the help of the tablet, because the ingredient is absorbed through the buccal mucosa directly into the blood stream and reaches the heart while the tablet gradually dissolves under the tongue. Otherwise, if the tablet would be swallowed, the active ingredient would face multiple degradation (stomach acids) and delay effects. Passing through the digestive system, it would reach the bloodstream after getting through the liver, therefore the ingredient would not reach the heart in time.

As disclosed in his patent U.S. Pat. No. 4,525,341 in 1984, Deihl found a more effective method than the sublingual tablet: the oral spray. The significance of the innovation is that the active ingredients compose a solution which is sprayed into the mouth. The fine mist gets on the buccal mucosa and is absorbed into the blood stream. With the help of his invention, Deihl uses not only the part of the buccal mucosa that is under the tongue, but the entire inner surface of the oral cavity as absorption area, therefore he increases bioavailability. As controlled tests show, the spray administered ingredients enter the blood stream through diffusion in 95 to 98 percent within 22 to 30 seconds which are very close results to the effectiveness of the above mentioned hospital procedures (intravenular, intramuscular, etc. forms) .

It can be assumed that the spray administration method which applies the absorption through the buccal mucosa is more effective in both absorption rate and onset rapidity than the conventional solid and liquid forms, moreover it can be used in a more simple and convenient way.

Liposomes and emulsions

Liposomes

Liposomes are microscopic particles with mean diameter of 20 to 100 nanometers that consist one or more lipid layers, enclosing .an aqueous core. The lipid layers are capable to merge into the cell membranes and subsequently allow transmission of the entrapped active ingredients through the membrane .

Liposomes were described first by A. D. Bangham and colleagues in 1965. They observed that liposomes tend to enclose part of their environment. The small liposome spheres are encircled by membrane bi-layers, just like the biological membranes surround the body cells. This remarkable resemblance lead to Bangham' s discovery. Today, we know what causes this similarity. The basic structure of biological membranes is made up by approximately the same ratio of lipids and proteins, in addition to them, there are 2 to 7 percent carbohydrates. Liposomes can be also regarded as models for biological membranes, however their structure is far more simple, they usually do not contain proteins, lipids are predominant. The most important lipids are phosphoglycerides in which the first and second carbon atoms of the glycerine are esterified with long carbon chain saturated and unsaturated fatty acids, the third carbon atom is bonded to an alcohol (ethanolamine, choline, glycerine or serine) through a phosphoric acid. The most important phosphoglycerides are phosphotidylcholine (lecithin) , phophotidylserine and phosphotidylinositol.

Liposomes are formed when the concentration of phospholipids is further increased. During this process, the bi-layer splits and the smaller parts that break down close themselves into it selves and form globes, so that bi-layer membrane enclosed particles, that is liposomes are created.

The use of liposomes as delivery systems for biologically active agents has become the subject of extensive research over the last ten years. Their nature and application in clinical practice are presented in numerous scientific publications, such as Barenholz, Amselem, Liposome Technology, 2. Edition, G. Gregoriadis, ed. , CRC press, 1992.

Liposomes may hold both hydrophilic and lipophilic substances, the technology of production has to be adjusted to the agents. One possibility is that the appropriate lipid mixture is dissolved with the help of an organic solvent and the solvent is eliminated by distillation. A thin lipid film remains on the wall of the test-tube, out of which the liposomes incorporating the lipophilic agents can be formed by dispersion in distilled water - by shaking or ultrasound irradiation. The lipophilic agents are added to the lipid components at the beginning of the preparation of the film, before the dissolution with the organic solvent, so that the agents can be incorporated into the liposome membrane.

Liposomes and the absorption through the buccal mucosa

The application of liposomes as delivery systems and the absorption of biologically active agents through the buccal mucosa were combined first by Keller as presented in his patent in 1999 (US Pat. No. 5,891,465). Although the description of the mentioned patent does not include it, however in the opinion of the author of the present invention, the great advantage of Keller's patented method was from the view of practical applicability the - following: Conventional solutions that are sprayed on the buccal mucosa enter the blood stream by diffusion. Diffusion is a passive delivery, through which particles start to move slowly from a region of high concentration (solution sprayed on the buccal mucosa) to one of lower concentration (cells of the buccal mucosa) . On the contrary, the lipids surrounding the liposomes go in between the lipid molecules of the cell membranes and pass on the agents they encapsulate to the cells: this method is active delivery, which is faster and more effective than diffusion.

A further advantage of the liposomes is that they are harmless to the skin and the human body, because the elements of this "delivery system" are identical to the basics that compose the biological membranes. This varied lipids are non-toxic, they don't initiate immune reaction and they decay biologically. Liposomes act as dosage systems as well, because their firm structure resolves gradually and the agents encapsulated are released slowly. Through the encapsulation, the agents in the liposomes are better protected against decomposition.

Disadvantage of the liposomes

However liposomes have the disadvantage of being able to encapsulate only a very small quantity of agents because of their small size, further it is difficult to produce them with the preferred stability, which has key importance in case of medications. The instability is both in vitro and in vivo significant: instability in vitro can manifest itself in different ways: often, the size and structure of the liposome particles show a discrepancy, the particles can accumulate, break up and release the biologically active hydrophilic agents.

In vivo the contact of liposomes with biological liquids often leads to an increased permeability of the liposome membrane. Especially, the hydrophilic agents encapsulated into the aqueous core can escape through diffusion. Usually, particles inside the liposomes strive for balance with the environment that surrounds the lipid layer of the liposome. In other words, only the quantity of hydrophilic agents can be hold in the liposome that is in equilibrium with the environment outside the liposome. If the substance outside becomes thinner - and this can happen because of contact with biological liquids, such as enzymes in the saliva, stomach acid or simple PH inconsistency,- the appropriate amount of the agents inside the ' liposome can leave by diffusion. The level of in vivo degradation of the liposomes varies by delivery method. Liposomes are ineligible for numerous delivery methods, such as water-based systems (drinks), because depending on its concentration, the solution can become turbid.

Emulsions , emulsifiers

Emulsions are mixtures of solutions that are naturally not miscible in each other, whereas the particles of one solution are dispersed in the other solution, for example water-in-oil or oil-in-water emulsions.

If materials that are insoluble in each other are mixed, they break up after short time and build two separate liquid parts. To achieve a stability of the emulsion, emulsifiers are needed, that is materials that reduce the surface tension in the liquid. Conventional emulsifiers are artificial additives, generally tensides, but also foamers and detergents that administered to the human body in higher dosage over a long time period can cause serious harm to the blood and blood-vessels.

OBJECT OF THE INVENTION, INDICATION OF THE PROBLEM TO BE SOLVED

Object of the present invention is to elaborate a composition for the delivery method of oral absorption, so that said composition incorporates a greater quantity of fat soluble agents than liposomes can do, and that said composition enables active delivery of the agents, just like liposomes do, but that said composition solves the problem of instability as seen at the liposomes, however without addition of synthetic emulsifiers. Aim of the inventor is to solve the problem in a way that is industrially applicable and economically executable.

SOLUTION OF THE PROBLEM

Nanoemulsion

Unlike emulsions, nanoemulsions do not contain synthetic emulsifiers. They deal with particles which surpass the particle size of the liposomes, they have diameters of 50-1000 nanometers, and in order to achieve stability they contain only naturally pure phosphotidylcholine. When producing nanoemulsions, phosphotidylcholine is made from lecithin which is the essential element of every single natural cell membrane. By means of high pressure technology phosphotidylcholine forms membranes which enclose the oil/fat droplets that are insoluble in water. In contrast to liposomes which have a water-soluble core, the particles of the nanoemulsions contain a fat-soluble core which is separated from the surrounding aqueous phase by one or more layers of phospholipids. As used herein, a nanoemulsion is a dispersion of nanoparticles with a lipid core in an aqueous surrounding, wherein said lipid core can be in both liquid or solid state.

The following chart gives a summary of the most important differences between conventional emulsions, liposomes and nanoemulsions:

Emulsions Nanoemulsions Liposomes

Definition dispersion of dispersion of solid dispersion of oil in water or liquid fats/lipids phospholipids in water in water

Core oil solid or liquid lipid water

Phospholipid content 0.5-2% 5-10% 0.1-5%

Non-natural emulsifier present none none

delivery capacity of fat soluble max. max. max. agents 10 mg/ml 100 mg/ml 20 mg/ ml

The main advantages of application of nanoemulsions are the following:

1. Nanoemulsions can deliver biologically active agents, both fat and water soluble: they can incorporate great amounts of fat soluble agents in their solid or liquid lipid core as well as and they can contain water soluble agents in the aqueous surrounding of their lipid core separated by phospholipids.

2. Alike liposomes, nanoparticles are bordered by phospholipid layers which allows active delivery. 3. Due to their bigger mean particle size, nanoemulsions are more stable than liposomes.

4. They do not contain non-natural, synthetic emulsifiers.

Preparation of nanoemulsions with high-pressure homogenisation

Nanoemulsions can be prepared by high-pressure homogenisation which can follow in a cold or hot process :

1. Similar to the production technology of liposomes, first the appropriate mixture of lipids and fat soluble biologically active agents have to be dissolved with the help of an organic solvent. The organic solvent is then removed by distillation.

2. The resulting lipid film is hydrated and dispersed by covering and shaking or ultrasound irradiation.

3. Third, high-pressure homogenisation is applied (up to 800 bar), for example with a Gaulin-type homogeniser (AVP Gaulin International, Holland) . High-pressure homogenisation is described in detail in Brandl, Liposome Technology, 2. Edition, G. Gregoriadis, Volume 1, Ch. 3, CRC Press, Boca Ranton, FI (1992) .

4. The particle size distribution of the formulation can be determined using an electronical microscope.

Examples

The following examples are intended to illustrate, but not limit the present invention.

Example 1 Multivitamin nanoemulsion spray formulation, 10 ml Additives %/ weight

Purified lecithin 1%/ weight Purified Water 95%/weight Natural flavouring agent 1%/weight

Rose hip extract 1% weight

Potassium sorbate 1%/weight

Biologically active agents weight

Vitamin E 10 mg

Folacin 200 meg

Vitamin C 60 mg

Vitamin B12 1 meg

Thiamin 1.4 mg

Biotin 150 meg

Niacin 18 m

Pantothenic acid 6 mg

Vitamin B6 2 mg

Beta-Carotene 5 mg

1. Lecithin and the fat soluble biologically active agents were dissolved in dichloromethane and the organic solvent was eliminated by distillation.

2. In a separate beaker, water and the water soluble biologically active agents were mixed.

3. The water mixture was added to the lipid mixture and mixed in a high speed homogenizing mixer at app. 15.000 rp for 5 minutes.

4. The solution was then mixed under high pressure at 800 bar for 10-15 cycles in a Microlab 70 Gaulin Homogenizing mixer.

5. The particle size of the nanoemulsion was controlled with the help of a N4MD Coulter particle-determination instrument (Coulter Electronics, Anglia) . The mean particle size was 90 +- 40 nanometer.

6. The flavouring agent, the rose hip extract and potassium sorbate was added and the solution was filled into the spray container.

The preparation process or the ingredients can be modified based on the experience and judgement of the professional supervising the procedure. Example 2 Nanoemulsion spray formulation for strengthening the immune system, 10 ml

Addi ives %/ weight

Purified lecithin 1 . 5% / weight

Purified water 75% /weight

Glycerine 15% weight

Natural flavouring agent 1% /weight

Potassium sorbate 1%/weight

Biologically active agents weight

Vitamin E 10 mg

Echinacea Purpurea

Augustifolia 15 mg

Hydras t is canadensys 4 mg

Example 3 Melatonin nanoemulsion spray formulation, 10 ml

Additives %/ weight

Purified lecithin 2%/ weight

Purified water 86%/weight

Cholesterol 0.2% weight

Glycerine 7.5%/weight Natural flavouring agent 1%/weight

Potassium sorbate 1%/weight

Biologically active agen ts %/ weight

Vitamin E 0.4% /weight

Melatonin 0.22%/weight

Claims

1. A nanoemulsion composition suitable for delivery of biologically active agents into the human body, said nanoemulsion is provided by a mechanical pump spray and administered though absorption into the blood stream through the buccal mucosa, wherein said biologically active agents can be one or more vitamins, minerals, amino acids, herbs, plant extracts, pharmaceutical agents, drugs or hormones.

2. The composition of claim 1, wherein said nanoemulsion has a mean particle diameter of 0,00005-0,0001 mm.

3. The composition of claim 1, wherein said nanoemulsion consists both water and fat soluble biologically active agents.

4. The composition of claim 1, wherein the particles of said nanoemulsion have a solid lipid core.

5. The composition of claim 1, wherein the particles of said nanoemulsion have a liquid lipid core.

6. The composition of claim 1, wherein said nanoemulsion does not contain non-natural emulsifiers.

7. The composition of claim 1, wherein said nanoemulsion contains pure phospholipid emulsifiers.

8. The composition of claim 1, wherein said nanoemulsion is administered by a mechanical pump spray into the oral cavity and absorbs through the buccal mucosa and reaches the blood stream.

9. The composition of claim 1, wherein said nanoemulsion contains one or more water soluble vitamins.

10. The composition of claim 1, wherein said nanoemulsion contains one or more fat soluble vitamins.

11. The composition of claim 1, wherein said nanoemulsion contains one or more minerals .

12. The composition of claim 1, wherein said nanoemulsion contains one or more amino acids .

13. The composition of claim 1, wherein said nanoemulsion contains coenzyme Q10.

14. The composition of claim 1, wherein said nanoemulsion contains one or more herbs or plant extracts, such as echinacea purpurea , gingko biloba , grape seed and pine bark proanthocyanidin extract, etc.

15. The composition of claim 1, wherein said nanoemulsion contains one or more pharmaceutical agents or drugs, such as glucosamine sulfate, chondroitin sulfate, etc.

16. The composition of claim 1, wherein said nanoemulsion contains one or more hormones, such as melatonin, DHEA, etc.